The overarching goal of the Developmental Glycobiology Unit is to determine the mechanisms by which protein O-glycosylation regulates basic biological processes to better understand the role of this modification in development and disease. Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs or GalNAcTs) that transfer the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted. Defects in this type of glycosylation are responsible for the human diseases familial tumoral calcinosis and Tn syndrome. Additionally, changes in O-glycosylation have been associated with tumor progression and metastasis. More recently, genome-wide association studies have identified the genes encoding the enzymes that are responsible for initiating O-glycosylation among those associated with HDL-cholesterol levels, triglyceride levels, congenital heart defects, colon cancer and bone mineral density. From these studies, it is apparent that this conserved protein modification has a multitude of biological roles. The focus of our research group is to elucidate the mechanistic role of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that there are at least 9 functional transferase genes in Drosophila and that at least one is required for viability. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Most recently, we have performed in vivo RNA interference (RNAi) to identify the remaining family members that are also essential for viability. We have discovered that 4 additional family members are required for viability. Specifically, RNAi to pgant4, pgant5, pgant7 or the putative glycosyltransferase CG30463 resulted in lethality. Additionally, these essential genes were required in specific tissues (mesoderm, digestive system and tracheal system), suggesting unique tissue-specific functions for each. Finally, we demonstrated that one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase resulted in reduced secretion of O-glycosylated proteins into the lumen of the gut and morphological alterations in the cells responsible for gut acidification. Mutations in this family member resulted in improper gut acidification. These studies have implications for the role of this protein modification in the proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. Following up on our Drosophila studies demonstrating a role for an O-glycosyltransferase in extracellular matrix (ECM) formation, we investigated whether the loss of a mammalian O-glycosyltransferase (Galnt1) has an effect on basement membrane formation and organogenesis using the murine submandibular gland (SMG) as a model system. The basement membrane of the developing SMG is a complex array of components that influence cell signaling, proliferation and differentiation; additionally, it is rich in O-glycosylated proteins. In these studies, we demonstrate that the loss of Galnt1 affects FGF-mediated cell proliferation during mammalian SMG organogenesis by influencing the secretion of basement membrane proteins. Mice deficient for the enzyme Galnt1 (that adds sugars to proteins during early stages of SMG development) resulted in intracellular accumulation of major BM components along with increased endoplasmic reticulum (ER) stress. Along with changes in BM composition, Galnt1 deficient glands displayed decreased FGF signaling, reduced AKT and MAPK phosphorylation, and reduced epithelial cell proliferation. Exogenous addition of BM component laminin to Galnt1 deficient glands rescued FGF signaling and the growth defects in a &#946;1-integrin-dependent manner. Our work demonstrated that O-glycosylation influences the composition of the secreted ECM during mammalian organ development, with resultant effects on cell signaling, proliferation and organ growth. These results highlight a conserved role for O-glycosylation in the establishment of cellular microenvironments and have implications for the role of this protein modification in both development and disease. Finally, we investigated the interplay between two different forms of glycosylation that are present on the same protein (alpha-dystroglycan). Specifically, we examined how the presence of O-mannosylation at specific sites on alpha- dystroglycan influences the subsequent addition of O-GalNAc by the GalNAcTs. By using a combination of enzymatic and mass spectroscopic methods, we found that the presence and specific location of O-mannose can result in either the regional exclusion or changes in the specific position of GalNAc addition. Our study demonstrates that one form of glycosylation can influence the presence and/or position of another form of glycosylation, suggesting that changes in both types of glycosylation may contribute to disease severity, as is commonly seen in muscular dystrophies. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of the studies described above will elucidate the mechanisms by which this conserved protein modification operates in both normal development and in disease susceptibility.